Serotonin is a neuromodulator capable of inducing and modulating a wide variety of behavioral functions such as sleep, appetite, locomotion, sexual activity and vascular contraction. It is accepted that serotonin activity is mediated by its interaction with receptors, designated serotoninergic receptors of 5-HT (for 5-hydroxytryptamine) receptors. Molecular biology studies as well as pharmacological studies have revealed the existence of a large number of subtypes of 5-HT receptors. The 5-HT receptors which have been described to date belong either to the family of receptors associated with ion channels (5-HT3 receptors), or to the family of receptors which interact with G proteins and which possess seven transmembrane domains. Moreover, analysis of the amino acid sequences has shown that the 5-HT receptors which interact with G proteins may be subdivided into six distinct groups: 5-HT1 receptors, comprising the mammalian subtypes 5-HT1a, 5-HT1b, 5-HT1d, 5-HT1e and 5-HT1f, as well as three Drosophila 5-HT receptors; 5-HT2 receptors comprising three subtypes, 5-HT2a, 5-HT2b and 5-HT2c; 5-HT4 receptor; 5-HT5 receptor; 5-HT6 receptor; and 5-HT7 receptor.
Drugs with affinity for 5-HT2 receptors are used to treat schizophrenia, Parkinsonism, and anxiety disorders. The 5-HT2c receptor subtype has been particularly interesting to investigators searching for the molecular bases of neuropsychiatric disorders. The 5-HT2c receptor is widely expressed in the brain where it is involved in regulating endocrine responses. Particular responses include the production and secretion of adrenocorticotropic hormone, oxytocin and prolactin. Genes for mouse, rat and human (Saltzman et al., Biochem. Biophys. Res. Commun. 181:1469, 1991) 5-HT2c receptors have been cloned. Burns et al., Nature vol. 387 15 May 1997 pp. 303-308 describes rat 5-HT2c receptor isoforms resulting from RNA editing events involving transcripts encodong the serotonin receptor. The functional state of 5-HT2c receptors in normal controls and various patient groups has been studied in vivo by administering 1-(3-chlorophenyl)piperazine (mCPP), a non-selective 5-HT2c agonist, and measuring hormonal and psychological responses. In alcoholism, panic disorder, seasonal affective disorder and obsessive-compulsive disorder, mCPP has been shown to induce different hormonal and psychological responses in patients and controls.
The invention includes mouse serotonin 5-HT2c receptor isoforms having amino acid replacements at one or more positions of the previously known mouse serotonin 5-HT2c receptor polypeptide sequence, specifically at one or more of positions 157, 159 and 161. The invention also includes isolated or purified isoforms, DNA encoding the isoforms, antibodies with specific binding to the receptor isoforms, assays for detecting the receptor isoforms, and expression vectors encoding the receptor isoforms.
The invention includes an isolated polypeptide comprising a sequence selected from the group consisting of (a) SEQ ID No. 3, (b) SEQ ID No. 5, (c) SEQ ID No. 7, (d) SEQ ID No. 9, (e) SEQ ID No. 11, and (f) fragments of (a), (b), (c), (d), and (e) wherein the polypeptide has serotonin activity. These polypeptides are mouse serotonin 5-HT2c receptor isoforms having amino acid replacements at one or more positions of the previously known mouse serotonin 5-HT2c receptor polypeptide sequence, specifically at one or more of positions 157, 159 and 161.
The polypeptides are useful for identifying ligands which bind with the serotonin 5-HT2c receptor and modulators of the serotonin 5-HT2c, and for identifying drugs with affinity for 5-HT2 receptors which are used to treat schizophrenia, Parkinsonism, and anxiety disorders.
The invention also includes a method for identifying modulators of a polypeptide comprising a sequence selected from the group consisting of (a) SEQ ID No. 3, (b) SEQ ID No. 5, (c) SEQ ID No. 7, (d) SEQ ID No. 9, (e) SEQ ID No. 11, and (f) fragments of (a), (b), (c), (d), and (e), comprising contacting in the presence of 5-HTa molecule or a mixture containing different molecules with a recombinant cell expressing at its surface a polypeptide of claim 1, under conditions permitting interaction between the polypeptide and 5-HT, and detecting molecules capable of modulating the activity of the polypeptide in the presence or absence of 5HT.
The invention also includes isloated nucleotide sequences coding for a polypeptide comprising a sequence selected from the group consisting of (a) SEQ ID No. 3, (b) SEQ ID No. 5, (c) SEQ ID No. 7, (d) SEQ ID No. 9, (e) SEQ ID No. 11, and (f) fragments of (a), (b), (c), (d), and (e), or the complementary strand, including such nucleotide sequences consisting of genomic sequences, cDNA sequences, RNA sequences, synthetic sequences and semi-synthetic sequences. Also within the the invention are such sequences placed under the control of signals permitting expression of the polypeptide in a host cell.
The invention also includes recombinant cells comprising nucleotide sequences of the invention, including eukaryotic and prokaryotic cells, and vectors for transforming the cells.
The invention also includes mammalian cell membranes comprising a polypeptide sequence selected from the group consisting of (a) SEQ ID No. 3, (b) SEQ ID No. 5, (c) SEQ ID No. 7, (d) SEQ ID No. 9, (e) SEQ ID No. 11, and (f) fragments of (a), (b), (c), (d), and (e) wherein the polypeptide has serotonin receptor activity. Optionally, the mammalian cell membranes are free of other receptor proteins. The mammalian cell membranes may be prepared from mammalian cells containing cDNA which encodes any of the polypeptides. The mammalian cells are transfected with recombinant DNA comprising vector DNA and cDNA which encode the polypeptides.
The invention also includes a method for identifying ligands of a polypeptide comprising a sequence selected from the group consisting of (a) SEQ ID No. 3, (b) SEQ ID No. 5, (c) SEQ ID No. 7, (d) SEQ ID No. 9, (e) SEQ ID No. 11, and (f) fragments of (a), (b), (c), (d), and (e), comprising contacting a molecule or a mixture containing different molecules with a recombinant cell expressing at its surface one of the polypeptides, under conditions permitting interaction between the polypeptide and the molecule, and detecting molecules bound to the polypeptide.
The invention also includes a method for identifying modulators of a polypeptide comprising a sequence selected from the group consisting of (a) SEQ ID No. 3, (b) SEQ ID No. 5, (c) SEQ ID No. 7, (d) SEQ ID No. 9, (e) SEQ ID No. 11, and (f) fragments of (a), (b), (c), (d), and (e), comprising contacting in the presence of 5-HTa molecule or a mixture containing different molecules with a recombinant cell expressing at its surface a polypeptide of claim 1, under conditions permitting interaction between the polypeptide and 5-HT, and detecting molecules capable of modulating the activity of the polypeptide in the presence or absence of 5HT.
The present invention further includes antibodies specific for the serotonin receptor isoform proteins of the invention which do not bind to the receptor as described by Yu et al. in GenBank X72230.
The term xe2x80x9cisolatedxe2x80x9d refers to material that is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
The mouse serotonin 5-HT2c subtype cDNA isoforms were subcloned into the expression vector pE3 which is modified as described below from pCEP4 (Invitrogen, Carlsbad, Calif.). Transient expression in HEK293e cells (Invitrogen, Carlsbad, Calif.) was accomplished by transfection of the cloned receptor cDNAs under the control of a CMV promoter into mammalian cells (e.g., HEK293e cells). Membranes prepared from the transfected cells are utilized for the determination of binding affinity, selectivity and specificity of the receptors for various ligands. Stable expression of the receptors in mammalian cells (e.g., CHO, HEK 293) is achieved after integration of the transfected cDNA into the chromosomes of the host cells. These stable cell lines constituently express the cloned receptors and can be propagated infinitely. Stable cell lines expressing the receptor cDNAs individually can be used in the binding assay to measure the affinity and selectivity of the receptors for serotonin agonists, antagonists and enhancers. These cells can also be used in functional assay to determine the agonist activity of any ligand.
Membranes prepared from transfected (HEK 293e) cells were utilized in a binding assay to measure the affinity of the receptors for radiolabeled agonists, e.g. [3H]5HT or [3H]mesulergine. The binding assay was performed by incubating membranes with increasing concentrations of radiolabeled agonists. Bound ligand was separated from free ligand by filtration. Bound radioactivity was measured by scintillation counting. Substances which bind to or enhance binding to expressed receptors in cells can be identified in competition binding assays with radiolabeled agonists. For the competition binding assay, membranes were incubated with a fixed concentration of radioligand and various concentrations of unlabeled agonists or antagonists.
Functional assay was performed by loading the receptor-expressing with [3H]inositol, incubating the cells with a ligand and measuring the accumulation of [3H]inositol trisphsophate inside the cells.
A transient expression system can be established by microinjection of in vitro transcribed mRNA from the cloned receptor cDNAs into Xenopus oocytes. The expression system allows measurement of the biological effects upon activation of the expressed receptors with ligand binding.
In the procedure for generating the isoforms of the present invention, transcripts encoding the mouse serotonin 5-HT2c receptor subtype undergo RNA editing events in which genomically encoded adenosine residues are converted to inosines by the action of double-stranded RNA adenosine deaminases (see Burns et al., Nature vol. 387 Ma. 15, 1997 pp. 303-308, which describes rat 5-HT2c receptor isoforms resulting from RNA editing events involving transcripts encodong the serotonin receptor). Sequence analysis of complementary DNA isolates from dissected mouse brain regions have indicated the tissue-specific expression of new receptor isoforms encoded by distinct RNA species. Editing of 5-HT2c receptor messenger RNAs alters the amino acid coding potential of the predicted second intracellular loop of the receptor and can lead to altered efficacy of the interaction between receptors and their G proteins. As shown in Table 3, the 5HT EC50 value is dependent on the amino acid sequence at position 157/159/161.
The table below compares a partial amino acid sequence of the published mouse serotonin 5-HT2c receptor (SEQ. ID No. 1) and the corresponding partial nucleotide sequence of the published mouse serotonin 5-HT2c receptor RNA, with amino acid sequences and nucleotide sequences corresponding to new isoforms of the mouse serotonin 5-HT2c receptor in the location of sequence variation. The isoforms have amino acid sequences identical to the published mouse serotonin 5-HT2c receptor polypeptide sequence except for specified amino acid replacements at one or more positions of the published mouse serotonin 5-HT2c receptor polypeptide sequence, specifically at one or more of positions 157, 159 and 161:
Alternative isoforms within the scope of the invention, otherwise identical to the natural mouse serotonin 5-HT2c receptor, have the following amino acid sequence and nucleotide sequence variations at positions 157, 159 and 161: